Engineering bacterium capable of efficiently expressing chitinase and plant growth promoting application

A high-efficiency expression technology of chitinase, applied in plant growth regulators, plant growth regulators, applications, etc., can solve the problem of low expression and activity of chitinase, and achieve the effect of promoting germination and growth

Active Publication Date: 2020-06-16
HUBEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies in the prior art, the present invention constructs an engineering bacterium that expresses chitinase efficiently through the gene

Method used

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  • Engineering bacterium capable of efficiently expressing chitinase and plant growth promoting application
  • Engineering bacterium capable of efficiently expressing chitinase and plant growth promoting application
  • Engineering bacterium capable of efficiently expressing chitinase and plant growth promoting application

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0044] Example 1. Construction of expression vector and protein expression

[0045] 1. Amplification of gene sequence

[0046] The primer pair (ChiA-F, ChiA-R) was designed according to the nucleotide sequence shown in SEQ ID NO.1.

[0047] Table 1: Primers for the amplification of chitinase-encoding genes

[0048]

[0049] Refer to Table 1. The underlined part of the forward primer is the restriction site of Cpo I, and the underlined part of the reverse primer is the restriction site of Not I. The sequence design of this site conforms to T 4 Sticky ends produced by DNA polymerase cutting.

[0050] PCR reaction system:

[0051]

[0052] PCR reaction conditions: 95°C pre-denaturation for 5min, 95°C denaturation for 30s, 56°C annealing for 30s, 72°C extension for 1min50s, 30 cycles of amplification, and finally 72°C for 10min extension.

[0053] Use 0.7% agarose gel electrophoresis to detect PCR products, and use DNA purification kit (Omega)

[0054] 2. Construction of recombinant expressio...

Example Embodiment

[0065] Example 2. Construction of cofactor genes and related vectors

[0066] 1. Cofactor gene amplification

[0067] According to the gene sequences of HAC1, ERV29, SEC16, COG5 and TRM1 cofactor derived from Pichia pastoris GS115, primers were designed for gene amplification, and these genes were constructed between the Xho I and Xba I restriction sites of pGAPZB vector.

[0068] See Table 2. The underlined part of the forward primer is the Xho I restriction site and its homologous sequence at the left end of the vector, and the underlined part of the reverse primer is the Xba I restriction site and its homologous sequence at the right end of the vector , The sequence design of this site conforms to T 5 The method of constructing vector with exonuclease.

[0069] Table 2: Primers for gene amplification of cofactor

[0070]

[0071] PCR reaction system:

[0072]

[0073] PCR reaction conditions: pre-denaturation at 98°C for 5min, denaturation at 98°C for 10s, annealing at 56°C for 5s, e...

Example Embodiment

[0078] Example 3. Co-expression of cofactor and 4 copies of chitinase recombinant strain

[0079] The plasmids pGAPZB-HAC1, pGAPZB-ERV29, pGAPZB-SEC16, pGAPZB-COG5, and pGAPZB-TRM1 were respectively digested with Avr II linearization, and the digested product was recovered from the solution. Then, after electrotransformation of GS115 competence of 4 copies of chitinase gene under the condition of 1.5kv, the YPD plate containing 100μg / mL Zeocin was coated and cultured at 28°C for 2 days. Then transfer the bacteria on the YPD (Zeocin) plate to the YPD plate and mark it. Then perform a yeast colony PCR to narrow the screening range. Take an appropriate amount of the selected bacteria and shake the flask with methanol to induce expression. Subsequent chitinase expression steps were performed according to the fifth point in Example 2 for methanol-induced expression.

[0080] The result is Figure 4 Shown: 1 is the chitinase 4-copy control, 2 / 3 / 4 / 5 / 6 is the chitinase when HAC1, ERV29,...

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Abstract

According to the engineering bacterium capable of efficiently expressing chitinase and the application of the engineering bacterium, the engineering bacterium takes pichia pastoris GS115 as an expression host, and strategies such as gene copy number, cofactor co-expression and high-density fermentation are combined, so that the obtained chitinase has the advantages of high expression quantity, high activity and the like. The chitinase expressed by the engineering bacterium can hydrolyze high-concentration colloidal chitin, the hydrolytic activity is good, the hydrolysate is mainly N-acetylglucosamine and then N, N-diacetyl chitobiose, and the chitinase can be used for promoting germination and growth of rice and wheat seeds.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an engineering bacterium for efficiently expressing chitinase and its application in the germination and growth of plant seeds. Background technique [0002] Chitin (chitin) is a linear polymer connected by N-acetyl-β-D-glucosamine through β-1,4-glycosidic bonds, and is one of the most abundant natural polymer compounds. The product of chitin hydrolysis by chitinase is chitooligosaccharide, which has high application value in the fields of medicine, health care and agriculture. Chitosan oligosaccharides are effective plant elicitors, which have the functions of inducing plant resistance and promoting plant growth. Chitin is widely found in the shells of various arthropods such as shrimps, crabs, and insects. In nature, as many as 10 chitins are synthesized by organisms every year. 11 tons, is one of the most abundant polysaccharides in nature. Biodegradat...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/56C12N9/42C12N15/81C12P19/26C12P19/14A01N43/16A01P21/00C12R1/84
CPCA01N43/16C12N9/2442C12N15/815C12P19/14C12P19/26C12Y302/01014
Inventor 张桂敏宋文贺妮莎张诺刘天罡
Owner HUBEI UNIV
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